ABSTRACT

The post-translational modifications (PTMs) phosphorylation and ubiquitylation regulate plasma membrane protein function. Here, we examine the interplay between phosphorylation and ubiquitylation of the membrane protein aquaporin-2 (AQP2) and demonstrate that phosphorylation can override the previously suggested dominant endocytic signal of K63-linked polyubiquitylation. In polarized epithelial cells, although S256 is an important phosphorylation site for AQP2 membrane localization, the rate of AQP2 endocytosis was reduced by prolonging phosphorylation specifically at S269. Despite their close proximity, AQP2 phosphorylation at S269 and ubiquitylation at K270 can occur in parallel, with increased S269 phosphorylation and decreased AQP2 endocytosis occurring when K270 polyubiquitylation levels are maximal. In vivo studies support this data, with maximal levels of AQP2 ubiquitylation occurring in parallel to maximal S269 phosphorylation and enhanced AQP2 plasma membrane localization. In conclusion, we demonstrate for the first time that although K63-linked polyubiquitylation marks AQP2 for endocytosis, site-specific phosphorylation can counteract polyubiquitylation to determine its final localization. Similar mechanisms might exist for other plasma membrane proteins.

INTRODUCTION

Phosphorylation and ubiquitylation are highly dynamic and regulated protein post-translational modifications (PTMs). Both modifications have the potential to mediate inducible protein–protein interactions and thereby regulate protein function. For membrane proteins, induction of phosphorylation, including poly-phosphorylation, is a mechanism for regulating the activity and intracellular trafficking of channels and transporters (Moeller et al., 2010; Rosenbaek et al., 2012; Tong et al., 2009; Yang et al., 2006). Ubiquitylation of membrane proteins through specific linkage is a common way of mediating internalization and promotion of protein degradation by the lysosomal pathway (Kazazic et al., 2009; Piper and Luzio, 2007; Vina-Vilaseca et al., 2011). ‘Cross-talking’ of phosphorylation and ubiquitylation has been proposed (Hunter, 2007), and phosphorylation often occurs as a priming event for ubiquitylation (Hunter, 2007; Murakami et al., 2009; Orford et al., 1997; Sehat et al., 2007). This occurs through a variety of mechanisms, such as phosphorylation-mediated regulation of the activity of E3-ligases, by generation of a phospho-E3-recognition (phosphodegron) site or by promoting localization of the protein to intracellular compartments where ubiquitylation can occur (Hunter, 2007). Although less frequent, and to our knowledge not reported for plasma membrane proteins, phosphorylation can mediate the opposite effect, for example, the phosphorylation of the kinase c-MOS inhibits ubiquitylation (Sheng et al., 2002). Alternatively, ubiquitylation can regulate protein phosphorylation by, for example, regulation of kinase activity (Hunter, 2007). Multiple PTMs in a protein are likely to act in concert rather than mediate regulatory effects alone. Such mechanisms could greatly increase fine tuning of protein function but also provide a great challenge in deciphering the complexity of protein regulation.

In this study, we examined the potential interplay between polyubiquitylation and poly-phosphorylation of the water channel aquaporin-2 (AQP2). AQP2 is expressed in the kidney collecting duct. Vasopressin-mediated accumulation of AQP2 in the apical plasma membrane of kidney principal cells is an essential event for urine concentration. The mechanism underlying membrane accumulation of AQP2 involves regulated insertion and removal of AQP2 from the plasma membrane (Brown, 2003; Knepper and Nielsen, 1993), a process that involves phosphorylation and ubiquitylation (Moeller et al., 2011). The C-terminal 15 amino acids of AQP2 contain a vasopressin-regulated poly-phosphorylated region with four phosphorylated serine residues at S256, S261, S264 and S269 (T in human) (Hoffert et al., 2006). These sites have differing roles for regulation of channel function and localization (see, for example, Fushimi et al., 1997; Hoffert et al., 2008; Kamsteeg et al., 2000; Moeller et al., 2011; Moeller et al., 2010; Nedvetsky et al., 2010; Tamma et al., 2011). It has been proposed that phosphorylation of AQP2 results in increased accumulation of AQP2 on the plasma membrane owing to reduced internalization (Lu et al., 2007; Moeller et al., 2010). The C-terminus of AQP2 is also subjected to polyubiquitylation at K270, with two or three ubiquitin molecules added through K63-linkage (Kamsteeg et al., 2006). Polyubiquitylation of AQP2 enhances its removal from the plasma membrane (Kamsteeg et al., 2006), similar to what has been reported for other proteins polyubiquitylated through K63-linkage (Haglund and Dikic, 2005).

Owing to the close proximity of AQP2 phosphorylation and ubiquitylation and the opposing roles of these PTMs for determining AQP2 localization, we utilized AQP2 as a model protein to examine the interplay between phosphorylation and ubiquitylation and whether one of these PTMs is always dominant to the other with respect to protein localization. We determined that although AQP2 S269 phosphorylation and K270 ubiquitylation can occur in parallel, the effects of phosphorylation can override the effect of K63-linked ubiquitylation on AQP2 endocytosis. Similar mechanisms might exist for other plasma membrane proteins.

RESULTS

The plasma membrane abundance of AQP2 is modulated by phosphorylation at S256 and S269

When the AQP2 mutants AQP2-S256D and AQP2-S269D are expressed in MDCK cells, they localize predominantly to the plasma membrane in basal conditions (Hoffert et al., 2008; Moeller et al., 2010; van Balkom et al., 2002). We and others (Rice et al., 2012) have also shown a decreased rate of AQP2 internalization in cells expressing single mutations at AQP2-S256D and AQP2-S269D compared to those expressing wild-type AQP2 (AQP2-WT); indicating that phosphorylation at both positions might be involved in retaining AQP2 in the plasma membrane. To dissect out the roles of individual sites we generated various polarized cell lines expressing double mutant AQP2 to examine whether AQP2-pS256 or AQP2-pS269 dominates in respect to AQP2 cellular localization. In cells expressing AQP2-S256A-S269A or AQP2-S256A-S269D, AQP2 was predominantly localized within the cell in basal conditions (Fig. 1), with greater colocalization with markers of the trans-Golgi network (TGN) (Vti1b and adaptin-G) and endosomes (EEA1) compared to WT-AQP2 (Fig. 2A and data not shown). In contrast, AQP2-S256D-S269A and AQP2-S256D-S269D showed predominantly membrane localization (Fig. 1), emphasized by very little colocalization with Vti1b or EEA1 (Fig. 2A). AQP2-S256D-S269D had significantly reduced colocalization with Vti1b and EEA1 compared to AQP2-S256D-S269A (Fig. 1; Fig. 2A). These results demonstrate that phosphorylation of AQP2 S256 is essential for AQP2 membrane localization, but the S256D-S269D form can accumulate on the plasma membrane more than the S256D-S269A form of AQP2. To confirm these results, we used a biotin-based internalization assay to examine AQP2 internalization rates in AQP2-S256D-S269A and AQP2-S256D-S269D double mutant cell lines. The AQP2-S256D mutation resulted in a decreased rate of AQP2 internalization compared to AQP2-WT (Fig. 2B). An additional decrease in internalization was observed when the AQP2-S269 residue was mutated to aspartic acid, suggesting that AQP2-S269-phosphorylation serves to further decrease AQP2 internalization when AQP2-S256 is phosphorylated.

Colocalization analysis of AQP2 with Vti1a and EEA1, and plasma membrane internalization rates of various AQP2 phosphorylation mutants. (A) Colocalization analysis was performed using Imaris software and Pearsons colocalization analysis followed by quantitative assessment. *P<0.05 compared with AQP2-WT; #P<0.05 between the indicated groups. n = 8 for each cell line. (C) Analysis of rates of internalization of AQP2 from the apical plasma membrane was based on biotinylation of surface proteins. Cells were treated with forskolin (25 µM) to accumulate AQP2 in the apical plasma membrane, followed by a forskolin washout period for various time points. *P<0.05 compared with AQP2-WT; #P<0.05 between AQP2-S256D-S269A and AQP2-S256D-S269D (n = 10).

AQP2 internalization is decreased in the presence of phosphatase inhibitors

Despite multiple attempts by our group (data not shown) and others (for example, Hoffert et al., 2008), a specific kinase that can modulate AQP2-S269 phosphorylation in vivo has not been identified. Thus, we made use of phosphatase inhibitors in an attempt to increase AQP2 phosphorylation specifically at individual sites and examine the effects of this inhibition on AQP2 plasma membrane accumulation. In the presence of the cell permeable phosphatase inhibitors okadaic acid (200 nM) and calyculin A (100 nM), following forskolin (an activator of adenylate cyclase resulting in increased AQP2 membrane localization) washout, AQP2 had a significantly decreased rate of internalization compared to controls (Fig. 3A). Concurrently with these effects on internalization, under the same conditions AQP2-S269 phosphorylation levels were significantly prolonged and enhanced (Fig. 3B), which supports a role for pS269 for decreasing AQP2 internalization. In contrast, there were no significant differences between control and treated groups when analyzing AQP2-pS256 levels, with levels of pS256-AQP2 remaining relatively constant throughout the internalization time period examined (Fig. 3C; supplementary material Fig. S3A). Although not significant from untreated controls, at the earliest time point examined during the washout period, there was a trend for increased AQP2-pS256 levels with okadaic acid and calyculin A, suggesting that AQP2-pS256 might be important for modulating AQP2 internalization at the early stages of endocytosis. Although there was a trend towards greater membrane abundance of AQP2 following forskolin treatment in the presence of inhibitors, the difference was not significant (Fig. 3D). Okadaic acid and calyculin A had no significant effect on AQP2-pS256 levels (Fig. 3E). AQP2-S269 phosphorylation was absent under control or upon okadaic acid or calyculin A treatment alone, significantly increased after forskolin treatment, and increased to a greater degree in the presence of phosphatase inhibitors (Fig. 3F). Taken together, these observations indicate that the effects of the phosphatase inhibitors on AQP2 internalization occur predominantly through modulation of AQP2-S269 phosphorylation. Additionally, AQP2-S269 phosphorylation was only detected during forskolin treatment and AQP2-pS269 was not observed in the internalized fraction (supplementary material Fig. S1A), suggesting rapid dephosphorylation of AQP2 at S269 during the internalization process.

Effects of the phosphatase inhibitors okadaic acid and calyculin A on AQP2 phosphorylation and internalization. (A) Internalized levels of AQP2 in MDCK cells at various time points after forskolin removal in the presence of phosphatase inhibitors okadaic acid (OA) and calyculin A (Cal-A), or in their absence (control). (B,C) Following pre-stimulation with forskolin, the effect of okadaic acid and calyculin A on phosphorylation levels of (B) AQP2-S256 and (C) AQP2-S269 during an internalization study. (D) The effect of calyculin A and okadaic acid on apical plasma membrane abundance of AQP2 following stimulation or not with forskolin (F). (E) The effect of calyculin A and okadaic acid on pS256-AQP2 levels. (F) The effect of calyculin A and okadaic acid on pS269-AQP2 levels. No pS269-AQP2 is detectable under control conditions or upon calyculin A and okadaic acid stimulation alone. *P<0.05 between treatment and control. NS, not significant. (G) Peptides representing AQP2-pS256 and AQP2-p269 were exposed to the catalytic subunits of PP1 and PP2A in vitro (units, u). Phosphospecific antibodies revealed that both phosphatases were able to dephosphorylate both positions S256 and S269.

The observed effects of okadaic acid and calyculin A on the phosphorylation status of AQP2 at S269 could be direct or indirect. At the concentrations utilized, the most likely phosphatases inhibited were PP1 or PP2A. AQP2-pS269 peptides were dephosphorylated by both PP1 and PP2A in vitro (Fig. 3G), indicating that a direct effect in our cell studies was plausible. However, in three independent experiments fostriecin, endothall or cantharidic acid (inhibitors of PP2A) did not significantly alter AQP2-S269 phosphorylation (supplementary material Fig. S1). Deltametrin, an inhibitor of PP2B, also did not affect AQP2-S269 phosphorylation (supplementary material Fig. S1), but mildly increased pS256 levels. These data indicate that PP1 is the likely phosphatase responsible for dephosphorylating AQP2-pS269 in vivo.

Comparison of AQP2 ubiquitylation and phosphorylation in various FLAG-tagged AQP2 mutants. (A) Pulldown of FLAG-tagged AQP2 in cells expressing wild-type FLAG-tagged AQP2 (F-AQP2-WT) and AQP2 with a mutation at K270 (F-AQP2-270R). Ubiquitylation is increased by compared to control (c), during forskolin treatment (f), after forskolin washout (wo), and for forskolin + TPA (f/tpa) treatment. Controls included were: (1) immunoprecipitation of cell lysates of non-FLAG-tagged AQP2 (AQP2-WT), (2) pure antibody in the absence of lysate (ab), and FLAG-tagged-AQP2 lysate without antibody (-ab). Ig hc indicates crossreactivity with IgG heavy chain. (B) Similar studies utilizing cell lines expressing FLAG-tagged AQP2-S269A and AQP2-S269D were performed. Alteration of the S269 site had no significant effect on AQP2 ubiquitylation. (C) Quantification from at least three independent immunoprecipitations (n = 6). Data were normalized relative to WT control. *P<0.05.

C-terminal phosphorylation and ubiquitylation as a general PTM crosstalk

Analysis of the ubiquitylation and phosphorylation datasets at PhosphoSite demonstrated that there are 95 other reported peptides (rat, mouse, human) that contain an ubiquitylated lysine residue at the penultimate position of the protein, similar to K270 of AQP2 (http://interpretdb.au.dk/database/Penultimate_Ubi.htm). Alignment of these sequences highlighted a preference for S, T or Y residues at the −1 position (relative to the K), and S and T residues at the −2 and −3 positions (supplementary material Fig. S3). These data indicate that phosphorylation and ubiquitylation often occur in close association with each other at the terminal tail of proteins and point towards a general PTM regulatory crosstalk.

In AQP2-WT cells, phosphorylation at AQP2-S269 significantly increased following forskolin treatment and decreased after wash-out. A similar pattern of phosphorylation was observed for cells expressing AQP2-K270R (Fig. 5A). Quantification from three independent experiments demonstrated that forskolin-induced AQP2-S269 phosphorylation in AQP2-K270R mutant cells was significantly higher than for AQP2-WT, but returned to similar baseline levels after washout (Fig. 5C). Similarly, although not as pronounced, AQP2-S256 phosphorylation levels were also significantly higher in AQP2-K270R mutant cells after forskolin stimulation (Fig. 5B).

AQP2 phosphorylation levels in MDCK cells expressing AQP2-WT or AQP2-K270R. Phosphorylation of pS256-AQP2 and pS269-AQP2 levels in MDCK cell lines expressing AQP2-WT and AQP2-K270R were determined by western blotting using phosphospecific antibodies. Treatment before preparation of gel samples included vehicle (Con), forskolin 25 µM for 30 min (For), or forskolin washout for 20 min (wo). (A) Representative western blots demonstrate AQP2-WT and AQP2-K270R phosphorylation levels. (B,C) Quantification from three independent experiments (n = 9) of the levels of pS256- and pS269-AQP2. *P<0.05 compared with control conditions or between groups as indicated.

Phosphatase inhibitor treatment increases ubiquitylation of AQP2

WT-AQP2 cells were subjected to forskolin or forskolin washout in the presence or absence of the phosphatase inhibitors okadaic acid and calyculin A (Fig. 6A). AQP2 ubiquitylation was greater under all conditions in the presence of phosphatase inhibitors, although this was only significant under control or forskolin conditions (Fig. 6B). AQP2-pS256 levels were not significantly higher under conditions where AQP2 ubiquitylation was increased (Fig. 6C). AQP2-S269 phosphorylation was only detected after forskolin treatment and was significantly higher in the presence of phosphatase inhibitors (Fig. 6D). Thus, phosphatase inhibitor treatment reduces AQP2 internalization (Fig. 3), and induces both ubiquitylation and phosphorylation of AQP2 at S269, two PTMs with opposing effects on membrane localization of AQP2.

Effects of the phosphatase inhibitors okadaic acid and calyculin A on AQP2 ubiquitylation and phosphorylation. FLAG-tagged AQP2 expressed in MDCK cells (AQP2-FLAG) was used for the study and non-FLAG-tagged AQP2 (AQP2 wt) was used as a negative control. Cell lysates were subjected to AQP2 pulldown by immunoprecipitation using anti-FLAG-antibody. Prior to pulldown the cells were treated with vehicle (Con), forskolin (For), and forskolin wash out (wo) in the absence or presence of the phosphatase inhibitors okadaic acid (OA) and calyculin A (CA) or the proteasome inhibitor MG-132. (A) Representative western blot of studies probed for ubiquitin, total AQP2 and phosphospecific antibodies against pS256-AQP2 and pS269-AQP2. Ig hc, IgG heavy chain. (B) Quantification of ubiquitylated AQP2 (Ubi-AQP2) relative to total AQP2 from three individual experiments (n = 6). (C) Quantification of phosphorylated AQP2 at S256 relative to total AQP2. (D) Quantification of phosphorylated AQP2 at S269 relative to total AQP2. *P<0.05 compared with control or between groups as indicated (n = 6).

Phosphorylation can reduce AQP2 internalization despite high levels of ubiquitylation

Assessment of AQP2 ubiquitylation versus membrane abundance and internalization rate was performed in a single study using AQP2-S256D-S269D cells, which had the lowest rate of AQP2 internalization (Fig. 1; Fig. 2A). Supporting our confocal imaging studies, AQP2-S256D-S269D was predominantly detected in the plasma membrane with no significant increase following forskolin stimulation (Fig. 7A). Ubiquitylation of AQP2-WT significantly increased following 15 min forskolin washout plus TPA treatment (Fig. 7B). In contrast, despite its greater plasma membrane abundance, AQP2-S256D-S269D had significantly greater ubiquitylation than AQP2-WT under control conditions and the levels of ubiquitylation were not increased by forskolin washout plus TPA treatment (Fig. 7B). In addition, despite the higher ubiquitylation levels, AQP2-S256D-S269D had a significantly lower degree of internalization from the apical plasma membrane (Fig. 7C). These data support a model whereby site-specific AQP2 phosphorylation can override the effect of K63-linked ubiquitylation on AQP2 endocytosis.

AQP2 phosphorylation can reduce AQP2 internalization despite high levels of ubiquitylation. Assessment of AQP2 ubiquitylation versus membrane abundance and internalization rate was performed in a single study using AQP2-S256D-S269D cells. (A) Levels of AQP2 in the apical plasma membrane under control or forskolin-stimulated conditions. AQP2-S256D-S269D was predominantly detected in the apical plasma membrane with no significant increase following forskolin stimulation. (B) Ubiquitylation of AQP2-WT significantly increased following 15 min forskolin washout+TPA treatment. In contrast, despite its greater apical plasma membrane abundance, AQP2-S256D-S269D had significantly greater ubiquitylation than AQP2-WT under control conditions and the levels of ubiquitylation were not increased by forskolin washout+TPA treatment. (C) Despite the higher ubiquitylation levels, AQP2-S256D-S269D had a significantly lower degree of internalization from the apical plasma membrane. #P<0.05 compared with control group, *P<0.05 between groups as indicated (n≥6).

In vivo modulation of AQP2 ubiquitylation by vasopressin

To examine whether AQP2 ubiquitylation could be regulated by vasopressin in vivo, AQP2 ubiquitylation levels were examined in kidney homogenates isolated from rats at various time points following treatment with the selective vasopressin type 2 receptor (AVPR2) agonist dDAVP (Fig. 8). A similar pattern of AQP2 ubiquitylation was observed in vivo as in our cell models. Supporting our cell model mechanisms of AQP2 retention mediated by phosphorylation at S269, maximum AQP2 ubiquitylation occurred 30 min after vasopressin stimulation, a time point when maximal levels of pS269-AQP2 were observed and AQP2 levels on the apical plasma membrane of collecting duct principal cells are the greatest (Moeller et al., 2009a).

AQP2 ubiquitylation is regulated by vasopressin in vivo. Rats were treated with saline (control) or dDAVP for 30 or 120 min, kidneys isolated and AQP2 immunoprecipitated. (A) Representative immunoblots demonstrating greatest levels of AQP2 ubiquitylation and pS269 levels 30 min following dDAVP treatment. All samples were compared on the same gel for quantitative analysis but, for viewing, lines indicate breaks in sample loading. (B) Summary of data from 16 rats (four per group). Ubiquitylated AQP2 levels were significantly higher than other groups at the 30-min timepoint. * P<0.05 compared with the 30-min control group.

DISCUSSION

Phosphorylation and ubiquitylation have been well studied for their roles in regulating protein function, yet crosstalk between the PTMs for regulation of plasma membrane proteins is not well defined. AQP2 undergoes both PTMs within a short span of C-terminal amino acids and thus is an ideal model to assess the interplay between phosphorylation and ubiquitylation. Previous studies have highlighted the role of AQP2-S256 phosphorylation for plasma membrane targeting of AQP2 (Fushimi et al., 1997; Hoffert et al., 2008; Katsura et al., 1997; McDill et al., 2006) and have suggested a special role for vasopressin-induced phosphorylation of AQP2 at S269 in plasma membrane accumulation (Hoffert et al., 2008; Moeller et al., 2009a; Moeller et al., 2009b; Moeller et al., 2010). Owing to the close association and interdependence of these two phosphorylation sites, it has proven to be difficult to understand the role of each individual site in the regulation of AQP2. In this study, we aimed to address this problem and, furthermore, suggest the possibility of a general cell biological phenomenon where site-specific phosphorylation of other plasma membrane proteins might override the endocytic effect of K63-linked ubiquitylation.

Both S256 and at S269 play roles in AQP2 membrane localization. When AQP2-S256 was mutated to aspartic acid, AQP2 was predominantly in the plasma membrane irrespectively of the status at the AQP2-S269 position. In contrast, preventing phosphorylation at residue AQP2-S256 resulted in intracellular localization of the protein regardless of the AQP2-S269 status. Examination of AQP2 internalization rates following agonist removal demonstrated that AQP2-S256D-S269A or AQP2-S256D-S269D mutants had slower internalization compared to AQP2-WT, with the AQP2-S256D-S269D form having the most prolonged period at the apical membrane. Combined with the reduced colocalization with markers of the endocytic system, these results strengthen the previously suggested role of the S269 site in modulating and/or prolonging AQP2 localization in the plasma membrane.

To investigate the isolated effect of AQP2-S269 phosphorylation in AQP2 membrane retention, we attempted to specifically inhibit de-phosphorylation of AQP2 at S256 or S269. In vitro de-phosphorylation assays demonstrated that PP1 and PP2A could dephosphorylate AQP2 at S256 or S269 in vitro. Calyculin A and okadaic acid in cell culture had no significant effect on pS256 levels following forskolin stimulation, but did greatly increase pS269 levels; this suggests that both PP1 and PP2A could play a role in AQP2 de-phosphorylation at S269. Selectively inhibiting PP2A or PP2B using endothall, fostriecin, cantharidic acid or deltamethrin did not result in significant changes in pS269 levels, suggesting that PP1 is the most likely protein phosphatase targeting pS269-AQP2 in vivo. Our results are in line with other studies that suggest a role for PP1 in regulation of AQP2 phosphorylation (Moeller et al., 2010; Zwang et al., 2009). A functional role for protein phosphatases in regulation of AQP2 has previously been suggested, although the effects of okadaic acid observed were attributed to a depolymerizing effect (disruption) on the actin cytoskeleton (Valenti et al., 2000).

Our studies reported here strengthen the hypothesis that vasopressin-induced AQP2-S269 phosphorylation mediates membrane retention of AQP2. In contrast, vasopressin removal (mimicked by forskolin washout) results in increased AQP2-K270 K63-linked ubiquitylation in the plasma membrane, which mediates enhanced AQP2 internalization (Kamsteeg et al., 2006). Thus, these neighboring sites have opposite effects on AQP2 subcellular localization. Owing to their close proximity we hypothesized that the two PTMs were linked, with enhanced AQP2-S269 phosphorylation reducing the levels of K270 ubiquitylation. However, relative to WT-AQP2, mutating AQP2-S269 to either alanine or aspartic acid had no significant effect on AQP2 ubiquitylation levels. Thus, it does not appear that AQP2-S269 phosphorylation directly regulates ubiquitylation. An alternative cross talk mechanism is that ubiquitylation regulates phosphorylation of AQP2. Mutation of AQP2-K270 to an arginine (preventing ubiquitylation) did not prevent forskolin-induced AQP2 phosphorylation but, in fact, facilitated AQP2-S269 phosphorylation. A possible explanation for this is that the presence of ubiquitin affects the binding or activity of the kinases involved in AQP2 phosphorylation (Hunter, 2007). Bioinformatics demonstrated that when a ubiquitylated lysine residue lies within the terminal three amino acids of a protein, there was a preference for S, T or Y residues at the −1 position (relative to the K). These data indicate that in other proteins, phosphorylation and ubiquitylation can occur in close association, suggesting the potential for a general PTM regulatory crosstalk mechanism.

As PTM of AQP2 at S269 and K270 can occur in parallel, we attempted to determine whether one of the modifications was dominant in terms of AQP2 membrane localization. Our data support a model whereby phosphorylation of AQP2 at S269 is able to override the internalization signal of neighboring K63-linked polyubiquitylation. This is supported by the following major findings. First, following forskolin stimulation and subsequent washout, in the presence of okadaic acid and calyculin A, there was a general increase in AQP2 ubiquitylation. However, this occurred with a paradoxical decrease in AQP2 internalization combined with prolonged and increased phosphorylation of S269. Second, AQP2-S269D cells had reduced AQP2 internalization rates despite the levels of AQP2 ubiquitylation being higher. Third, in AQP2-S256D-S269D cells AQP2 was predominantly on the plasma membrane under control conditions, despite 2-fold–3-fold greater levels of AQP2 ubiquitylation. Fourth, in AQP2-S256D-S269D cells AQP2 had significantly reduced internalization rates despite constitutively high levels of AQP2 ubiquitylation. Finally, in vivo, at a time point when AQP2 plasma membrane levels are greatest, AQP2 ubiquitylation and pS269 phosphorylation levels are also greatest. To our knowledge, this is the first report that phosphorylation can sequester a protein in the plasma membrane despite the protein being marked for internalization by K63-linked ubiquitylation. What is the basis for such a mechanism (summarized in supplementary material Fig. S4)? The first possibility is centered on previous observations (Lu et al., 2007; Moeller et al., 2009b) that phosphorylation of AQP2 decreases its interaction with proteins of the ‘endocytic machinery’. It is feasible that, even when marked for internalization by K63-linked ubiquitylation, phosphorylation of AQP2 sequesters the protein in a membrane compartment that is not susceptible to clathrin-mediated endocytosis, the major pathway for AQP2 internalization (Brown and Orci, 1983; Brown et al., 1988; Sun et al., 2002). Such AQP2 ‘endocytic-resistant domains’ have been proposed earlier by Brown and colleagues (Bouley et al., 2006). Alternatively, phosphorylation of AQP2 might alter the plasma membrane dynamics of AQP2 between raft and non-raft compartments, and indirectly affect the rate at which ubiquitylated AQP2 can be processed for clathrin-mediated endocytosis. The final mechanism that we propose is centered on the molecular recognition of the ubiquitylated AQP2 by ubiquitin-binding domain (UBD)-containing proteins, e.g. epsin (Horvath et al., 2007; Hurley and Wendland, 2002). It is plausible that phosphorylation of AQP2 alters the affinity of UBD proteins for AQP2, thus affecting clathrin pit formation and endocytosis. Furthermore, epsins have low affinity to single ubiquitin moieties and multiple ubiquitin moieties are required to form an efficient internalization signal (Piper and Luzio, 2007). As we cannot determine how many ubiquitin moieties are present within the AQP2 tetramer formed on the surface, it is possible that the stoichiometry of phosphorylation versus ubiquitylation within each tetramer in the plasma membrane might be a factor that determines the internalization rate of AQP2.

In conclusion, our data suggest that site-specific phosphorylation of AQP2 can override the effect of K63-linked ubiquitylation on AQP2 endocytosis. We propose that the effects of closely associated phosphorylation and ubiquitylation that we observe for AQP2 could be a general mechanism for other plasma membrane proteins and act as a mechanism to fine-tune protein localization and function.

MATERIALS AND METHODS

Transfection and cell culture

Mutant forms of AQP2 and N-terminal FLAG-tagged AQP2 were generated by site-directed mutagenesis. Generation of stable MDCK cell lines and cell culture conditions were as previously described (Hoffert et al., 2008). Multiple individual cell lines were individually characterized by examination of cell morphology, and AQP2 expression by western blotting, immunocytochemistry and RT-PCR. For all experiments, cells were cultured on semi-permeable supports (0.4 µM pore size, Corning) until a confluent monolayer formed. mpkCCDc14 cells were cultured on filters as described previously (Yu et al., 2009) until the trans-epithelial resistance (TER) was above 5 kOhm/cm2. Subsequently dDAVP (Sigma, 10−9 M) was added in serum-free medium to the basolateral compartment for 4 days to induce AQP2 expression.

Antibodies and chemicals

Affinity-purified rabbit phospho-specific antibodies against pS256-AQP2 and pS269-AQP2 or against total AQP2 upstream of phosphorylation sites have previously been characterized (Hoffert et al., 2008; Moeller et al., 2009a; Nishimoto et al., 1999). Mouse anti-ubiquitin (P4D1) was from Cell Signaling and rabbit anti-FLAG antibody (F7425) was from Sigma-Aldrich. The phosphatase inhibitors cantharidic acid, okadaic acid, endothall, deltamethrin, fostreicin and calyculin A were from Calbiochem. Forskolin (Sigma) was used at a final concentration of 25 µM. Intracellular marker antibodies were purchased from BD Transduction Laboratories and used for immunocytochemistry at: mouse-anti EEA1, 1∶100 (early endosome marker), Vti1b, 1∶500 (marker of post-Golgi vesicle trafficking), and adaptin G, 1∶500 (marker of late Golgi and the trans-Golgi network and/or endosomes).

Immunoblotting

Standard procedures were utilized and blots were developed using ECL detection. Quantification was performed on non-saturated films by determining signal intensity in each band using Quantity One 4.2.3 software densitometry analysis.

Immunocytochemistry

Confocal microscopy and colocalization analysis

A Leica TCS SL confocal microscope with an HCX PL APO 63× oil objective lens (numerical aperture 1.40) was used for obtaining image stacks with a z-distance of 0.1 µm between images. Images were obtained at room temperature. Microscope settings (PMT offset and gain, sampling period, and averaging) were identical. To quantify the degree of colocalization, a minimum of four independent stacks from two different experiments were obtained sequentially by using a 488-nm laser line and emission between 505 and 540 nm for Alexa Fluor 488 and a 546-nm laser line and emission over 585 nm for Alexa Fluor 546 and 555. Background correction and quantification of colocalization was performed using the colocalization function of Imaris image analysis software.

Phosphatase inhibitor studies and internalization assays

Cells were washed twice in pure medium and pre-incubated for 30 mins in the presence of inhibitors or vehicle. In the presence of inhibitors or vehicle, cells were stimulated with forskolin (25 µM) for 20 min at 37°C. After three washes on ice in ice-cold PBS-CM, pH 8.0 (PBS containing 1 mM CaCl and 0.1 mM MgCl2), cells were incubated for various time points in medium containing vehicle or inhibitors. For some studies, gel samples were prepared directly after two washes in ice-cold PBS-CM by addition of sample buffer containing DTT. For internalization studies, the cells were surface biotinylated after the forskolin treatment followed by re-incubation at 37°C in medium containing inhibitors or vehicle for various timepoints. MesNa-based stripping of surface biotin and internailization assays were performed as previously described (Moeller et al., 2010). All experiments were performed on two wells of cells at least three times.

Immunoprecipitation using MDCK cells

For immunoprecipitations using phosphatase inhibitors (okadaic acid and calyculin A) cells were incubated in control or forskolin-containing medium for 30 min in the presence or absence of inhibitors. For studies indicating ‘washout’, forskolin was removed, and cells were washed and re-incubated for 20 min in presence or absence of inhibitors. For other studies, cells were pretreated as annotated for 30 min followed by ‘washout’ in either control medium, or medium containing forskolin (25 µM) or forskolin and 0.1 µM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 15 mins. Samples were lysed in 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.25% Na-Deoxycholate, 1% Triton X-100, 1 mM EDTA, 20 mM N-ethylmelamide, containing protease inhibitors Leupeptin and Phefa-block (Boehringer Mannheim) and phosphatase inhibitor cocktail tablets (PhosSTOP, Roche Diagnostics A/S). Following sonication, samples were centrifuged at 10,000 g for 10 min at 4°C. Samples were assayed for protein concentration and immunoprecipitation was performed at 4°C for 1 h using ∼150 µg of lysate and 1 µg of FLAG antibody in a total volume of 500 µl. Lysates were subsequently incubated with 20 µls of Protein-A–agarose (Santa Cruz Biotechnology) followed by washing three times with lysis buffer and elution in sample buffer. Specificity of AQP2 ubiquitylation was confirmed by performing immunoprecipitations following full denaturation of cell lysates in SDS-buffer (0.5% SDS, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0) before heating to 65°C for 10 min (data not shown).

In vivo assessment of AQP2 ubiquitylation

All animal protocols comply with the European Community guidelines for the use of experimental animals and were performed in accordance to licenses for the use of experimental animals issued by the Danish Ministry of Justice. Before each experiment, rats had free access to standard rat chow and water. 16 male wistar rats (200 g) were randomly separated into experimental or control groups. Rats were injected intramuscularly with 10 ng dDAVP in saline (or saline alone) and killed after 30 or 120 min by cervical dislocation. Kidneys were homogenized in 5% sorbitol, 5 mM histidine-imidazole, and 0.5 mM Na2EDTA containing phosphatase and protease inhibitors and 20 mM N-ethylmelamide. Homogenates were mixed 1∶1 with 2× lysis buffer 100 mM Tris-HCl pH 7.4, 300 mM NaCl, 0.5% Na-Deoxycholate, 2% Triton X-100, 2 mM EDTA, 20 mM N-ethylmelamide, containing protease inhibitors Leupeptin and Phefa-block (Boehringer Mannheim) and phosphatase inhibitor cocktail tablets (PhosSTOP, Roche Diagnostics A/S). IP was performed as for MDCK cells.

Statistics

Data was tested for normal distribution using the D'Agostino-Pearson omnibus test and Graphpad Prism Software. Data fitting a normal distribution were analyzed using one-way ANOVA followed by Bonferroni's Multiple Comparison Test. Multiple comparisons tests were applied only when a significant difference (P<0.05) was determined in the ANOVA. Data not fitting a normal distribution were assessed using a nonparametric Kruskal–Wallis test. All data are presented as mean±s.e.m.

Footnotes

Competing interests

The authors declare no competing interests.

Author contributions

H.B.M.O. developed and designed the study, performed the experiments, analyzed data, wrote the manuscript; T.A. and J.S.H. performed the experiments; T.P. performed the experiments and assisted in the development of the study; R.A.F. developed and designed the study, performed the experiments, analyzed data and wrote the manuscript. All authors approved the final manuscript.

Funding

This work was supported by the Danish Medical Research Council; The Lundbeck Foundation; the Kidney Association (Nyreforeningen); the Novo Nordisk Foundation; the Carlsberg Foundation; and the Aarhus University Forsknings Fond. TP is currently supported by CU Research Cluster: 2014 Ratchadapisek Sompoch Endowment Fund, Chulalongkorn University.

(2009b). Role of multiple phosphorylation sites in the COOH-terminal tail of aquaporin-2 for water transport: evidence against channel gating.Am. J. Physiol.296, F649–F657.doi:10.1152/ajprenal.90682.2008

Pavan Vedula and Anna Kashina propose a new concept, the actin code, which encompasses the regulation of the essential functions of mammalian actins at the nucleotide level, rather than at the level of amino acids.

“To me, there are no real boundaries between chemistry, biology, physics and maths.”

Tony leads a group at the Mechanobiology Institute in Singapore, focusing on dissecting the structure–function relationship that underlies protein complexes that are involved in cell migration and adhesion. He shares his thoughts on why you don’t necessarily have to choose between the different branches of science that you find fascinating.

We also feature interviews with first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Check out our recent First Person interview with Julia Abitbol.

The second in our series of cell dynamics meetings now turns to organelles. This May 2019 meeting in Lisbon, Portugal, aims to bring together scientists studying the interface between organelles and the cytoskeleton at different scales and perspectives using a range of model systems. Find out more and register your interest here.

We are currently seeking proposals for four Workshops to be held in 2020. Do you have an idea for a Workshop? Please let us know and you could be one of our 2020 Workshop organisers. You focus on the science, we focus on the logistics. We are particularly keen to receive proposals from postdocs. Deadline date for applications is 25 May 2018.

Meet the preLighters! In the latest interview with our preLights community, the preLights team caught up with James Gagnon, Assistant Professor at the University of Utah, to talk about his research, how science can be made more open, his enthusiasm for the preLights project and the fun sides of being a junior PI.

Alexander García Ponce investigated how hematopoietic stem cell (HSC) cross the vascular wall and reach the bone marrow as part of his PhD project, with an aim to improving the outcome of HSC transplantation for individuals with leukaemia. A Travelling Fellowship from JCS allowed Alexander to advance his research at the Sanquin Blood Bank in The Netherlands. Read more on his story here.

Where could your research take you? Join Alexander and apply for the next round of Travelling Fellowships from JCS by 25 May 2018.